Canine Strangles Case Reveals a New Host Susceptible to Infection with Streptococcus equi
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《微生物临床杂志》
Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, United Kingdom
University of Cambridge, Department of Veterinary Medicine, Cambridge CB3 0ES, United Kingdom
ABSTRACT
We report the first documented case of canine strangles due to infection with Streptococcus equi in a dog with enlarged lymph nodes. Genetic typing, via sequencing of 12 housekeeping genes and the SeM gene, demonstrated the isolate to be a member of a common equine strain type circulating in the United Kingdom.
CASE REPORT
A 7-year-old male (neutered) golden retriever was referred to the Soft Tissue Surgery Service at the Centre for Small Animal Studies, Animal Health Trust, for investigation of laryngeal swelling and lethargy. As a similar episode due to an infection following the entry of a foreign body had occurred 5 months previously and had responded to antibiotics, a similar infection was suspected.
On clinical examination, a firm, irregular, 6-cm-diameter swelling was noted in the area of the right submandibular lymph node. Clinical examination was otherwise unremarkable. A hematology profile revealed a marked mature neutrophilia (29 x 109/liter; reference range, 4 x 109 to 12 x 109/liter). On the biochemistry profile, creatinine kinase was mildly elevated at 189 IU/liter (reference range, 21 to 56 IU/liter).
Oral examination under general anesthesia demonstrated bilateral tonsillar inflammation. Thoracic radiographs were within normal limits. Ultrasound examination of the neck revealed four distinct masses, the largest of which measured 2 by 2 by 4 cm; the others ranged from 1 to 3 cm. The three small masses had hyperechoic medullas and were compatible with enlarged lymph nodes. The larger mass was hyperechoic in relation to the other masses, but again, an enlarged lymph node was the primary differential diagnosis. A magnetic resonance imaging scan of the mandibular area confirmed that the swelling was due to submandibular and retropharyngeal lymphadenopathy combined with diffuse soft tissue swelling, which was centered on the right tonsil and extended caudally to the submandibular lymph nodes. No foreign body was identified.
Biopsy of the laryngeal swelling by the referring veterinary practice revealed chronic active inflammation of skeletal muscle and fat with edematous granulation tissue. A fine-needle aspirate of the submandibular lymph node was consistent with lymphoid hyperplasia and mild neutrophilic lymphadenitis. Core biopsies of the submandibular swelling under ultrasound guidance were nondiagnostic. The right submandibular lymph node was therefore biopsied by excision of the largest of the previously described masses and submitted for anaerobic and aerobic bacterial culture, fungal culture, and histopathology. The right tonsil was also biopsied. Pending the histopathological and microbiological results, the dog was prescribed clavulanate-amoxicillin (15 mg/kg of body weight twice daily) and carprofen (3 mg/kg once daily). A good response to the medication was noted, with a marked decrease in the lymphadenopathy over the following 5 days.
Following overnight fixation, biopsy samples collected in 10% buffered formalin were embedded in paraffin wax, and 5-μm sections were cut, mounted on glass slides, and stained with hematoxylin and eosin using standard techniques. Histopathological examination of biopsy tissue from the right tonsil revealed extensive follicular hyperplasia of the submandibular lymphoid tissue. Multifocal to coalescing infiltrates of neutrophils and lesser numbers of plasma cells were present within the tonsillar parenchyma, with dense accumulations of neutrophils present exocytosing through the overlying pharyngeal epithelium. Samples of the right submandibular lymph node revealed marked fibrosis of the lymph node capsule and parenchyma and moderate to marked hyperplasia of lymphoid follicles. The histological features of these samples were indicative of a subacute to chronic neutrophilic lymphadenitis of the right tonsil and tissue repair, secondary to a previous lymphadentitis, within the submandibular lymph node.
Microbiological analysis of tonsillar tissue identified the presence of beta-hemolytic streptococci (strain 8229) on COBA streptococcal selective agar (bioMerieux). The strain was identified as Streptococcus equi by a lack of fermentation of trehalose, ribose, and sorbitol in Purple broth (Becton Dickinson). A series of genetic tests were performed to determine if the isolate represented a novel subtype of S. equi.
DNA was isolated as previously described (7), and S. equi genes were PCR amplified using the primers listed in Table 1 with Vent DNA polymerase (New England Biolabs) and 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The PCR products were purified on QIAquick spin columns (QIAGEN), and the sequences of both strands of the PCR fragments were determined using an ABI3100 DNA sequencer with BigDye fluorescent terminators and the primers used in the initial PCR amplification.
Sequence data were assembled using SeqMan 5.03 (DNAstar Inc.), and high-quality double-stranded-sequence data were used for further analysis. Each of the 12 housekeeping gene sequences (fba, murI, tdk, arcC, nrdE, gmk, spi, recA, yqiL, tpi, ddlA, and pros) from the canine isolate were used to perform BLASTN analyses against the assembled online S. equi strain 4047 genome-sequencing database at http://www.sanger.ac.uk/Projects/S_equi/. This revealed that strain 8229 was identical to strain 4047 across all 12 loci, in agreement with other studies that have demonstrated minimal diversity of S. equi isolates (from horses) using multilocus enzyme electrophoresis or multilocus sequence typing of S. equi (6; C. Robinson, unpublished results). A novel strain-typing method, based upon sequence variation of the SeM gene, has recently been developed enabling discrimination between S. equi isolates (2, 7). SeM gene sequence data were submitted to http://pubmlst.org/szooepidemicus/seM/. This determined that strain 8229 had a SeM allele 6. PCR analysis also confirmed the presence of the genes encoding the pyrogenic mitogens, SePE-H, SePE-I, SpeL, and SpeM, found in equine strains of S. equi (1, 3, 8).
The clinical and pathological findings of the enlarged tonsil and submandibular lymph nodes were consistent with a chronic bacterial infection. The anatomical location of the lesions indicated that the most likely source of the infection was the oropharyngeal cavity. This is consistent with the finding of S. equi within the tonsillar tissue.
Sequence-typing methods determined that strain 8229 was indistinguishable from the most prevalent subtype of S. equi circulating in United Kingdom horses, which was implicated in 9 of 27 strangles outbreaks (7). Taken together, these data suggest that this dog was infected with an S. equi isolate of equine origin and that strain 8229 probably does not represent a new lineage of S. equi.
S. equi is believed to have evolved from an ancestral strain of Streptococcus zooepidemicus, and the two organisms share 97% sequence identity. In dogs, S. zooepidemicus infection is typically associated with increased severity of canine infectious respiratory disease (4), canine pneumonia (5), and septicemia (9). However, this is the first case of disease in dogs that has been attributed to infection with S. equi.
S. equi is the causative agent of strangles, one of the most commonly diagnosed and important infectious diseases of horses worldwide. The disease is characterized by pyrexia, followed by profuse nasal discharge and abscessation of the lymph nodes of the head and neck. The swelling of the lymph nodes in the head and neck may, in severe cases, restrict the airway, and it is this clinical feature that gave the disease "strangles" its name.
The dog lived on premises where horses were kept and stabled, and given the sequencing information, in-contact horses represent the most likely source of infection. However, the owners maintain that they have never had a case of equine strangles on their premises, so the actual source of this infection remains undetermined.
We hypothesize that this case may have implications for the control of S. equi infections in horse stables. It is possible that colonization of the oropharyngeal tracts of dogs in contact with horses may provide a previously unrecognized reservoir of the infection. Therefore, the significance of S. equi-infected dogs in contact with horses in the control of S. equi infection among horses warrants further study.
ACKNOWLEDGMENTS
We thank the referring veterinary surgeon, N. McLeod, Bishops Stortford Veterinary Hospital, United Kingdom.
FOOTNOTES
REFERENCES
Alber, J., A. El-Sayed, S. Estoepangestie, C. Lammler, and M. Zschock. 2005. Dissemination of the superantigen encoding genes seeL, seeM, szeL and szeM in Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Vet. Microbiol. 109:135-141.
Anzai, T., Y. Kuwamoto, R. Wada, S. Sugita, T. Kakuda, S. Takai, T. Higuchi, and J. F. Timoney. 2005. Variation in the N-terminal region of an M-like protein of Streptococcus equi and evaluation of its potential as a tool in epidemiologic studies. Am. J. Vet. Res. 66:2167-2171.
Artiushin, S. C., J. F. Timoney, A. S. Sheoran, and S. K. Muthupalani. 2002. Characterization and immunogenicity of pyrogenic mitogens. Microb. Pathog. 32:71-85.
Chalker, V. J., H. W. Brooks, and J. Brownlie. 2003. The association of Streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease. Vet. Microbiol. 95:149-156.
Garnett, N. L., R. S. Eydelloth, M. M. Swindle, S. L. Vonderfecht, J. D. Strandberg, and M. B. Luzarraga. 1982. Hemorrhagic streptococcal pneumonia in newly procured research dogs. J. Am. Vet. Med. Assoc. 181:1371-1374.
Jorm, L. R., D. N. Love, G. D. Bailey, G. M. McKay, and D. A. Briscoe. 1994. Genetic structure of populations of beta-haemolytic Lancefield group C streptococci from horses and their association with disease. Res. Vet. Sci. 57:292-299.
Kelly, C., M. Bugg, C. Robinson, Z. Mitchell, N. Davis-Poynter, J. R. Newton, K. A. Jolley, M. C. Maiden, and A. S. Waller. 2006. Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks. J. Clin. Microbiol. 44:480-486.
Proft, T., P. D. Webb, V. Handley, and J. D. Fraser. 2003. Two novel superantigens found in both group A and group C Streptococcus. Infect. Immun. 71:1361-1369.
Sundberg, J. P., D. Hill, D. S. Wyand, M. J. Ryan, and C. H. Baldwin. 1981. Streptococcus zooepidemicus as the cause of septicemia in racing greyhounds. Vet. Med. Small Anim. Clin. 76:839-842.(Jane Ladlow, Timothy Scas)
University of Cambridge, Department of Veterinary Medicine, Cambridge CB3 0ES, United Kingdom
ABSTRACT
We report the first documented case of canine strangles due to infection with Streptococcus equi in a dog with enlarged lymph nodes. Genetic typing, via sequencing of 12 housekeeping genes and the SeM gene, demonstrated the isolate to be a member of a common equine strain type circulating in the United Kingdom.
CASE REPORT
A 7-year-old male (neutered) golden retriever was referred to the Soft Tissue Surgery Service at the Centre for Small Animal Studies, Animal Health Trust, for investigation of laryngeal swelling and lethargy. As a similar episode due to an infection following the entry of a foreign body had occurred 5 months previously and had responded to antibiotics, a similar infection was suspected.
On clinical examination, a firm, irregular, 6-cm-diameter swelling was noted in the area of the right submandibular lymph node. Clinical examination was otherwise unremarkable. A hematology profile revealed a marked mature neutrophilia (29 x 109/liter; reference range, 4 x 109 to 12 x 109/liter). On the biochemistry profile, creatinine kinase was mildly elevated at 189 IU/liter (reference range, 21 to 56 IU/liter).
Oral examination under general anesthesia demonstrated bilateral tonsillar inflammation. Thoracic radiographs were within normal limits. Ultrasound examination of the neck revealed four distinct masses, the largest of which measured 2 by 2 by 4 cm; the others ranged from 1 to 3 cm. The three small masses had hyperechoic medullas and were compatible with enlarged lymph nodes. The larger mass was hyperechoic in relation to the other masses, but again, an enlarged lymph node was the primary differential diagnosis. A magnetic resonance imaging scan of the mandibular area confirmed that the swelling was due to submandibular and retropharyngeal lymphadenopathy combined with diffuse soft tissue swelling, which was centered on the right tonsil and extended caudally to the submandibular lymph nodes. No foreign body was identified.
Biopsy of the laryngeal swelling by the referring veterinary practice revealed chronic active inflammation of skeletal muscle and fat with edematous granulation tissue. A fine-needle aspirate of the submandibular lymph node was consistent with lymphoid hyperplasia and mild neutrophilic lymphadenitis. Core biopsies of the submandibular swelling under ultrasound guidance were nondiagnostic. The right submandibular lymph node was therefore biopsied by excision of the largest of the previously described masses and submitted for anaerobic and aerobic bacterial culture, fungal culture, and histopathology. The right tonsil was also biopsied. Pending the histopathological and microbiological results, the dog was prescribed clavulanate-amoxicillin (15 mg/kg of body weight twice daily) and carprofen (3 mg/kg once daily). A good response to the medication was noted, with a marked decrease in the lymphadenopathy over the following 5 days.
Following overnight fixation, biopsy samples collected in 10% buffered formalin were embedded in paraffin wax, and 5-μm sections were cut, mounted on glass slides, and stained with hematoxylin and eosin using standard techniques. Histopathological examination of biopsy tissue from the right tonsil revealed extensive follicular hyperplasia of the submandibular lymphoid tissue. Multifocal to coalescing infiltrates of neutrophils and lesser numbers of plasma cells were present within the tonsillar parenchyma, with dense accumulations of neutrophils present exocytosing through the overlying pharyngeal epithelium. Samples of the right submandibular lymph node revealed marked fibrosis of the lymph node capsule and parenchyma and moderate to marked hyperplasia of lymphoid follicles. The histological features of these samples were indicative of a subacute to chronic neutrophilic lymphadenitis of the right tonsil and tissue repair, secondary to a previous lymphadentitis, within the submandibular lymph node.
Microbiological analysis of tonsillar tissue identified the presence of beta-hemolytic streptococci (strain 8229) on COBA streptococcal selective agar (bioMerieux). The strain was identified as Streptococcus equi by a lack of fermentation of trehalose, ribose, and sorbitol in Purple broth (Becton Dickinson). A series of genetic tests were performed to determine if the isolate represented a novel subtype of S. equi.
DNA was isolated as previously described (7), and S. equi genes were PCR amplified using the primers listed in Table 1 with Vent DNA polymerase (New England Biolabs) and 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The PCR products were purified on QIAquick spin columns (QIAGEN), and the sequences of both strands of the PCR fragments were determined using an ABI3100 DNA sequencer with BigDye fluorescent terminators and the primers used in the initial PCR amplification.
Sequence data were assembled using SeqMan 5.03 (DNAstar Inc.), and high-quality double-stranded-sequence data were used for further analysis. Each of the 12 housekeeping gene sequences (fba, murI, tdk, arcC, nrdE, gmk, spi, recA, yqiL, tpi, ddlA, and pros) from the canine isolate were used to perform BLASTN analyses against the assembled online S. equi strain 4047 genome-sequencing database at http://www.sanger.ac.uk/Projects/S_equi/. This revealed that strain 8229 was identical to strain 4047 across all 12 loci, in agreement with other studies that have demonstrated minimal diversity of S. equi isolates (from horses) using multilocus enzyme electrophoresis or multilocus sequence typing of S. equi (6; C. Robinson, unpublished results). A novel strain-typing method, based upon sequence variation of the SeM gene, has recently been developed enabling discrimination between S. equi isolates (2, 7). SeM gene sequence data were submitted to http://pubmlst.org/szooepidemicus/seM/. This determined that strain 8229 had a SeM allele 6. PCR analysis also confirmed the presence of the genes encoding the pyrogenic mitogens, SePE-H, SePE-I, SpeL, and SpeM, found in equine strains of S. equi (1, 3, 8).
The clinical and pathological findings of the enlarged tonsil and submandibular lymph nodes were consistent with a chronic bacterial infection. The anatomical location of the lesions indicated that the most likely source of the infection was the oropharyngeal cavity. This is consistent with the finding of S. equi within the tonsillar tissue.
Sequence-typing methods determined that strain 8229 was indistinguishable from the most prevalent subtype of S. equi circulating in United Kingdom horses, which was implicated in 9 of 27 strangles outbreaks (7). Taken together, these data suggest that this dog was infected with an S. equi isolate of equine origin and that strain 8229 probably does not represent a new lineage of S. equi.
S. equi is believed to have evolved from an ancestral strain of Streptococcus zooepidemicus, and the two organisms share 97% sequence identity. In dogs, S. zooepidemicus infection is typically associated with increased severity of canine infectious respiratory disease (4), canine pneumonia (5), and septicemia (9). However, this is the first case of disease in dogs that has been attributed to infection with S. equi.
S. equi is the causative agent of strangles, one of the most commonly diagnosed and important infectious diseases of horses worldwide. The disease is characterized by pyrexia, followed by profuse nasal discharge and abscessation of the lymph nodes of the head and neck. The swelling of the lymph nodes in the head and neck may, in severe cases, restrict the airway, and it is this clinical feature that gave the disease "strangles" its name.
The dog lived on premises where horses were kept and stabled, and given the sequencing information, in-contact horses represent the most likely source of infection. However, the owners maintain that they have never had a case of equine strangles on their premises, so the actual source of this infection remains undetermined.
We hypothesize that this case may have implications for the control of S. equi infections in horse stables. It is possible that colonization of the oropharyngeal tracts of dogs in contact with horses may provide a previously unrecognized reservoir of the infection. Therefore, the significance of S. equi-infected dogs in contact with horses in the control of S. equi infection among horses warrants further study.
ACKNOWLEDGMENTS
We thank the referring veterinary surgeon, N. McLeod, Bishops Stortford Veterinary Hospital, United Kingdom.
FOOTNOTES
REFERENCES
Alber, J., A. El-Sayed, S. Estoepangestie, C. Lammler, and M. Zschock. 2005. Dissemination of the superantigen encoding genes seeL, seeM, szeL and szeM in Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Vet. Microbiol. 109:135-141.
Anzai, T., Y. Kuwamoto, R. Wada, S. Sugita, T. Kakuda, S. Takai, T. Higuchi, and J. F. Timoney. 2005. Variation in the N-terminal region of an M-like protein of Streptococcus equi and evaluation of its potential as a tool in epidemiologic studies. Am. J. Vet. Res. 66:2167-2171.
Artiushin, S. C., J. F. Timoney, A. S. Sheoran, and S. K. Muthupalani. 2002. Characterization and immunogenicity of pyrogenic mitogens. Microb. Pathog. 32:71-85.
Chalker, V. J., H. W. Brooks, and J. Brownlie. 2003. The association of Streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease. Vet. Microbiol. 95:149-156.
Garnett, N. L., R. S. Eydelloth, M. M. Swindle, S. L. Vonderfecht, J. D. Strandberg, and M. B. Luzarraga. 1982. Hemorrhagic streptococcal pneumonia in newly procured research dogs. J. Am. Vet. Med. Assoc. 181:1371-1374.
Jorm, L. R., D. N. Love, G. D. Bailey, G. M. McKay, and D. A. Briscoe. 1994. Genetic structure of populations of beta-haemolytic Lancefield group C streptococci from horses and their association with disease. Res. Vet. Sci. 57:292-299.
Kelly, C., M. Bugg, C. Robinson, Z. Mitchell, N. Davis-Poynter, J. R. Newton, K. A. Jolley, M. C. Maiden, and A. S. Waller. 2006. Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks. J. Clin. Microbiol. 44:480-486.
Proft, T., P. D. Webb, V. Handley, and J. D. Fraser. 2003. Two novel superantigens found in both group A and group C Streptococcus. Infect. Immun. 71:1361-1369.
Sundberg, J. P., D. Hill, D. S. Wyand, M. J. Ryan, and C. H. Baldwin. 1981. Streptococcus zooepidemicus as the cause of septicemia in racing greyhounds. Vet. Med. Small Anim. Clin. 76:839-842.(Jane Ladlow, Timothy Scas)